Identifying Mechanisms of Aurora Kinase A in Centrosome Clustering Using Chemical Genetics

利用化学遗传学鉴定中心体聚类中 Aurora 激酶 A 的机制

• 批准号: 10321536
• 负责人: Gina M Tomarchio
• 金额: $ 4.68万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-11 至 2023-12-10
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10321536
• 关键词: Address; Alleles; Aneuploidy; Binding; Biological Assay; CRISPR/Cas technology; Cells; Centrosome; Chromosome Segregation; Chromosomes; Complex; Data; Daughter; Defect; Development; Dimerization; Diploid Cells; Dynein ATPase; Ensure; Genomic Instability; Hematologic Neoplasms; Hematology; Human; Impairment; In Vitro; Influentials; Kinesin; Lead; Literature; Malignant Neoplasms; Masks; Mediating; Microscopy; Microtubule-Organizing Center; Microtubules; Mitosis; Mitotic; Mitotic Spindle Apparatus; Mitotic spindle; Motor; Movement; Mutation; Pathway interactions; Patients; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Positioning Attribute; Prognosis; Proteins; Publishing; Role; Sirolimus; Sister Chromatid; Site; Solid; Somatic Cell; System; Testing; Total Internal Reflection Fluorescent; Transgenic Organisms; Work; analog; attenuation; aurora kinase A; base; cancer cell; cancer survival; cancer therapy; cell cortex; cell motility; chemical genetics; chromosome missegregation; daughter cell; design; dynactin; experimental study; inhibitor; insight; interest; mutant; overexpression; phosphoproteomics; prevent; scaffold; small molecule inhibitor; spatiotemporal; targeted cancer therapy; tool; tumorigenesis

SUMMARY ABSTRACT Mitotic kinases and phosphatases are essential for preventing errors in chromosome segregation that can lead to aneuploidy and cancer. Aurora Kinase A (AurA) is an essential mitotic kinase that is often upregulated in human cancers. AurA has a well-studied role in assembly of the mitotic spindle apparatus, a complex microtubule array responsible for separating sister chromatids into two identical daughter cells. It is challenging to study AurA function outside of spindle assembly because inhibition causes disruption of the mitotic spindle which can mask other relevant phenotypes. Further, most AurA small molecule inhibitors can be promiscuous. We have developed a system that circumvents these issues by allowing for specific and conditional inhibition of AurA activity. Using cultured human cells, we have inactivated endogenous AurA with CRISPR-Cas9 and rescued its activity with a genetically modified, transgenic AurA allele (AurA-AS). The genetic alteration makes this allele specifically sensitive to modified ATP competitive inhibitors that cannot inhibit any other cellular kinases. Our objective is to use this tool to study the connection between AurA and cancer. Specifically, we are interested in the connection between AurA and centrosome number amplifications, which are common in both solid and hematological malignancies. Extra centrosomes can create mitotic spindles with more than two poles thus causing devastating chromosome mis-segregation. AurA inhibition has been associated with formation of multipolar spindles in the literature. Our preliminary data suggests that AurA activity prevents this multipolar spindle phenotype by promoting the “clustering” of extra centrosomes into two spindle poles. This led us to our central hypothesis that AurA phosphorylation is essential for bi-polar spindle formation in the presence of extra centrosomes. Based on evidence in the literature, we hypothesize that AurA mediates centrosome clustering via phosphorylation of spindle-pole focusing kinesin HSET (Aim 1) and via promoting localization of mitotic spindle positioning factor NuMA to the cell cortex (Aim 2). To determine if AurA-phosphorylation of HSET is important for efficiency of centrosome clustering, we will use in vitro kinase assays to show direct phosphorylation (1.1). We will use HSET phospho-site mutants to determine if this phosphorylation is relevant to centrosome clustering (1.2) and to perform in vitro microtubule motility assays to determine how AurA phosphorylation effects HSET’s activity (1.3). Further, we will use microscopy to determine if loss of AurA impairs mitotic spindle positioning or NuMA localization to the cell cortex (2.1) and if altered NuMA localization effects efficiency of centrosome clustering (2.2). All proposed experiments use our established system for studying AurA. This work is of particular interest as both solid and hematological malignancies often have increased number of centrosomes; by clustering them into two spindle poles, they can prevent division of their chromosomes into multiple, inviable daughter cells. This work will describe a connection between AurA and a unique cancer survival mechanism and will therefore be informative for design and implementation of AurA-specific cancer therapies.

摘要 摘要 有丝分裂激酶和磷酸酶对于防止染色体分离错误至关重要，这些错误可能导致 极光激酶 A (AurA) 是一种重要的有丝分裂激酶，在细胞中经常上调。 AurA 在有丝分裂纺锤体（一种复杂的微管）的组装中具有经过充分研究的作用。 负责将姐妹染色单体分离成两个相同子细胞的阵列 研究 AurA 具有挑战性。 在纺锤体组件之外发挥作用，因为抑制会导致有丝分裂纺锤体的破坏，从而掩盖 此外，大多数 AurA 小分子抑制剂可能是混杂的。 开发了一种系统，通过允许 AurA 的特定和条件抑制来规避这些问题 使用培养的人类细胞，我们用 CRISPR-Cas9 灭活了内源性 AurA 并拯救了它。 具有转基因 AurA 等位基因 (AurA-AS) 的活性 基因改变使得该等位基因。 对不能抑制任何其他细胞激酶的修饰 ATP 竞争性抑制剂特别敏感。 目标是使用这个工具来研究 AurA 和癌症之间的联系。具体来说，我们感兴趣的是。 AurA 和中心体数量扩增之间的联系，这在固体和中心体中都很常见 血液恶性肿瘤。额外的中心体可以产生具有两个以上极的有丝分裂纺锤体。 破坏性的染色体错误分离与 AurA 的形成有关。 我们的初步数据表明 AurA 活动可以阻止这种多极纺锤体。 通过促进额外中心体“聚集”成两个纺锤体极来形成纺锤体表型，这导致了我们的研究。 中心假设是，AurA 磷酸化对于存在额外的双极纺锤体形成至关重要 根据文献中的证据，我们追踪 AurA 通过介导中心体聚类。 纺锤体极聚焦驱动蛋白 HSET 的磷酸化（目标 1）并通过促进有丝分裂纺锤体的定位 将因子 NuMA 定位到细胞皮质（目标 2）以确定 HSET 的 AurA 磷酸化是否重要。 为了提高中心体聚类的效率，我们将使用体外激酶测定来显示直接磷酸化 (1.1)。 我们将使用 HSET 磷酸位点突变体来确定这种磷酸化是否与中心体聚类相关 (1.2) 并进行体外微管运动以确定 AurA 测定磷酸化如何影响 HSET 此外，我们将使用显微镜来确定 AurA 的丢失是否会损害有丝分裂纺锤体的定位或。 NuMA 定位到细胞皮层 (2.1) 以及 NuMA 定位是否影响中心体的效率 聚类（2.2）。所有提出的实验都使用我们建立的系统来研究 AurA。 引起兴趣的是实体瘤和血液恶性肿瘤通常都会增加中心体的数量； 将它们聚集成两个纺锤体极，可以防止染色体分裂成多个不可生存的 这项工作将描述 AurA 与独特的癌症生存机制之间的联系。 因此，将为 AurA 特异性癌症疗法的设计和实施提供信息。